concanavalin-a and Granulomatous-Disease--Chronic

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.

Topics: Blotting, Western; Cell Membrane; Concanavalin A; Cytoplasm; Granulomatous Disease, Chronic; GTP-Binding Proteins; Humans; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophils; Phosphoproteins; rac GTP-Binding Proteins; Tetradecanoylphorbol Acetate

In order to characterize the mechanism by which the anti-rheumatic gold complex auranofin (AF) affects the functions of resting and activated polymorphonuclear leukocytes (PMN) the following studies were performed: (1) The effect of AF on the major processes involved in the respiratory burst of PMN: glucose transport and phosphorylation; hexose monophosphate (HMP) shunt activity in intact cells and in a cell-free system; superoxide production by particulate fractions and intact PMN measured as lucigenin-dependent chemiluminescence. (2) A comparison of the effects of AF added to the PMN before, at the time of, or subsequent to the stimulants [N-formyl-methionyl-leucyl phenylalanine (FMLP), concanavalin A (ConA), calcium ionophore (A23187) and phorbol myristate acetate (PMA)]. (3) The effect of AF on PMN activated by two stimulates (PMA, ConA) added sequentially. AF (0.1-10 microM) caused a dose-dependent inhibition of lucigenin-dependent chemiluminescence regardless of the activator (FMLP, ConA, A23187, PMA) when AF was added before the activator. In contrast, when AF was added to PMN after stimulation, it inhibited only the chemiluminescence of PMN stimulated by PMA. Furthermore, the chemiluminescence was largely unaffected by AF in sequentially activated PMN. The relative sensitivity to AF of the various processes studied indicates that blockade of the activation signal appears to be responsible for inhibition of the respiratory burst of PMN.

Topics: Auranofin; Cell Survival; Concanavalin A; Deoxyglucose; Granulomatous Disease, Chronic; Humans; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Oxygen; Pentose Phosphate Pathway; Signal Transduction; Stimulation, Chemical; Superoxides; Tetradecanoylphorbol Acetate

Human neutrophils, triggered by Concanavalin A, were cytotoxic against chicken red blood cell targets as determined by the 51Cr release method. The cytolysis increased with the effector: target ratio, reaching optimal levels when 2-4 neutrophils were available for each chicken red blood cell. The target cell lysis required an optimal release of highly reactive oxygen by-products by neutrophils, since neutrophils from a patient with chronic granulomatous disease failed to exhibit any cytolytic activity. Superoxide dismutase, catalase and inhibitors of heme-containing peroxidases (azide and cyanide) significantly inhibited the neutrophil-mediated cytotoxicity. Together these results indicate that superoxide anion and the myeloperoxidase-hydrogen peroxide system are simultaneously involved in the target cell injury by Concanavalin A-triggered neutrophils.

Topics: Adult; Child; Chromium Radioisotopes; Concanavalin A; Cytotoxicity, Immunologic; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Male; Neutrophils; Peroxidase; Superoxides

Topics: Adult; Aging; Child; Concanavalin A; Fetal Blood; Granulomatous Disease, Chronic; Humans; Immunity, Cellular; Infant, Newborn; Lectins; Luminescent Measurements; Luminol; Male; T-Lymphocytes, Cytotoxic; Time Factors

Using a previously described luminol-dependent photometric chemiluminescence (CL) assay we have investigated the relative significance of the free radicals in the CL phenomenon associated with the respiratory burst of granulocytes and monocytes. The O-2 scavenger, superoxide dismutase, quenches approximately 50% of CL emission from resting and stimulated cells of both types. CL production from granulocytes and monocytes, in the presence of catalase, indicates that H2O2 plays a much less significant role in monocyte light emission than in that of granulocytes. Sodium azide, an 1O2 scavenger and potent inhibitor of peroxidase, and sodium benzoate, an OH. scavenger, both induced 90% reductions of light output from both cell types in resting or stimulated states. The distinct effects of cytochalasins on granulocytes and monocytes further suggest distinct CL generating mechanisms for each cell type. No difference was observed between granulocyte and monocyte CL response in chronic granulomatous disease (CGD) and other clinically related but unknown phagocyte metabolic disorders, whereas selective CL response abnormalities were observed in patients with severe isolated phagocyte chemotaxis defects.

Topics: Concanavalin A; Cytochalasins; Cytotoxicity, Immunologic; Free Radicals; Granulocytes; Granulomatous Disease, Chronic; Humans; Luminescent Measurements; Methylmannosides; Monocytes; Phagocytosis

We have studied the pattern of membrane binding site redistribution, movement, and reappearance in polarized and nonpolarized human neutrophils using fluorescein and rhodamine-labeled lectins as probes. In suspension, polymorphonuclear leukocytes (PMN) were spherical and displayed a random array of recognition sites for all of the probes. PMN polarized in suspension by 10(-6) M N-formyl-L-methionyl-L-phenylalanine (f-Met-Phe), and PMN attached to substrate accumulated the bound lectin recognition site complex at the uropod (for Con A; 92.0 +/- 0.2% of cells and 91.3 +/- 9.8% of cells, respectively). Glutaraldehyde fixation of neutrophils oriented in a chemotactic gradient prior to lectin addition revealed the innate unbound recognition site array. Unbound Con A recognition sites were clustered at the front of 74.7 +/- 0.8% of cells in a "headlight" pattern, but binding sites for other lectins were distributed randomly around the polarized cell. When bound Con A complexes are swept to the tail of the polarized living PMN, "new" unbound Con A binding sites appear at the front of the cell. Neither cycloheximide nor KCN nor colchicine interferred with new binding site appearance. Cytochalasin B and sodium iodacetate prevented PMN polarization and interfered with appearance of new receptors. This suggests that these fresh sites are uncovered, previously cryptic binding sites rather than newly synthesized structures. Lectin binding site topography and movement are related to the functional state of the PMN. Since both Con A and certain bacteria bind to mannose derivatives, we postulate that the "headlight pattern" and uncovering of fresh binding sites aid the PMN in engulfing organisms as the phagocyte moves forward.

Topics: Cell Membrane; Concanavalin A; Granulomatous Disease, Chronic; Humans; Lectins; Microscopy, Fluorescence; Neutrophils; Receptors, Mitogen

Stimulation of normal granulocytes with chemotactic factor, phorbol myristate acetate, concanavalin A, and calcium ionophore results in rapid depolarization which precedes the 'respiratory burst'. Treatment of granulocytes in chronic granulomatous disease with these stimulants fails to generate chemiluminescence. This defect is associated with an absence of transmembrane potential shifts in response to treatment with chemotactic factor, phorbol myristate acetate, and concanavalin A while depolarization in response to A23187 is unaffected by this disease state.

Topics: Calcimycin; Chemotactic Factors; Concanavalin A; Granulocytes; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Membrane Potentials; Tetradecanoylphorbol Acetate

Topics: Animals; Azides; Benzoates; Catalase; Chickens; Concanavalin A; Cytotoxicity, Immunologic; Erythrocytes; Granulomatous Disease, Chronic; Histidine; Humans; Lectins; Male; Mannitol; Neutrophils; Oxygen; Peroxidase; Proteins; Superoxide Dismutase

A simple quantitative method to measure release of hydrogen peroxide and superoxide anions from leukocytes using less than 10 microliter blood is described. Whole blood instead of a leukocyte suspension was used. The release of hydrogen peroxide was measured fluorometrically by adding a blood sample to an assay mixture containing homovanillic acid, peroxidase and azide. The release of superoxide anions was measured spectrophotometrically with a dual wavelength spectrophotometer by adding a blood sample to an assay mixture containing cytochrome C. The normal values of the releasing activities are given.

Topics: Concanavalin A; Cytochalasins; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Kinetics; Leukocytes; Oxygen; Spectrophotometry; Superoxides

In an effort to restore oxidant-dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsone derivative, 4-amino-4'-hydroxylaminodiphenyl sulphone (DDS-NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mM DDS-NOH for 30 min, the rate of glucose-1-14C oxidation via hexose monophosphate (HMP) shunt increased 2--4-fold and that of iodination of ingested zymosan particles 1.5--2.7-fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS-NOH improved in vitro killing of Staph. aureus. DDS-NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexes suggesting interference by the drug with microtubule-associated function. Thus, optimal concentrations of DDS-NOH may be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.

Topics: Aminobiphenyl Compounds; Blood Bactericidal Activity; Catalase; Concanavalin A; Dapsone; Glucose; Granulomatous Disease, Chronic; Humans; Immunologic Capping; Neutrophils; Superoxide Dismutase

Topics: Concanavalin A; Cytochalasins; Cytochrome c Group; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Neutrophils; Phagocyte Bactericidal Dysfunction; Superoxides

The role of surface -SH groups on the hexose monophosphate pathway activity in resting human polymorphonuclear leucocytes was studied. Endotoxin, taurocholic acid and concanvalin A stimulated [I-14C]glucose oxidation to a greater degree than [6-14C]glucose oxidation. The stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid and concanavalin A could be prevented by p-chloromercurybenzene sulphonic acid, an inhibitor of surface -SH groups. In contrast, polymorphonuclear leucocytes from four patients with chronic granulomatous disease failed to show stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid or concanavalin A.

Topics: 4-Chloromercuribenzenesulfonate; Biological Transport; Blood Glucose; Cell Membrane Permeability; Colchicine; Concanavalin A; Endotoxins; Granulomatous Disease, Chronic; Hexosephosphates; Humans; Lectins; Neutrophils; Sulfhydryl Compounds; Surface Properties